D. Kapoor

Gene cloning, expression and homology modeling of hemolysin gene from Aeromonas hydrophila.

Hemolysin is a significant toxin secreted by Aeromonas hydrophila, which contributes pathogenicity of fish to humans. The complete ORF of hemolysin gene (1886 bp) was amplified using PCR. It was cloned in TA and sub-cloned in pET28a vector then transformed into Escherichia coli BL21(DE3) codon plus RP cells expressed by the induction with 1.0 mM of IPTG. The expected size of expressed protein was 68.0 kDa estimated by migration in 12% SDS-PAGE. Anti-His monoclonal antibodies were used to substantiate the recombinant protein by Western blotting. The percent similarity between hemolysin of A. hydrophila with other hemolytic toxins revealed that the hemolysin/aerolysin/cytotoxin sequence varied from 99.35 to 50.40%. Homology modeling was used to construct 3-D structure of hemolysin of A. hydrophila with the known crystal 3-D structure (PDB: 1XEZ). This protein can be used for immunoassays and it is suitable for vaccine candidate against A. hydrophila infection.

Detection of aerolysin gene in Aeromonas hydrophila isolated from fish and pond water

Aerolysin is a hemolytic toxin encoded by aerolysin gene (1482 bp) that plays a key role in the pathogenesis of Aeromonas hydrophila infection in fish. New speciesspecific primers were designed to amplify 326 bp conserved region of aerolysin gene for A. hydrophila. Twenty-five isolates of A. hydrophila recovered from fish and pond water were studied for detection of aerolysin gene. Aerolysin gene was detected in 85% of the isolates during the study. The designed primers were highly specific and showed no cross reactivity with Escherichia coli, Aeromonas veronii, Vibrio cholerae, Flavobacterium spp., Chyseobacterium spp. and Staphylococcus aureus. The sensitivity limit of primers for detection of aerolysin gene in the genomic DNA of A. hydrophila was 5 pg.

Development of monoclonal antibodies to rohu [Labeo rohita] immunoglobulins for use in immunoassays.

Serum immunoglobulins [Ig] of rohu [Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [+/-SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [+/-2.34] and 51.84% [+/-2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [+/-1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.

Production of monoclonal antibodies specific to major outer membrane protein of Edwardsiella tarda

Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and gastro and extra-intestinal infections in humans. Monoclonal antibodies (MAbs) were produced against outer membrane proteins (OMPs) of E. tarda ET-7, isolated from diseased snakehead (Ophiocephalus punctatus). Two stable hybridoma clones, designated as 3F10 and 2C3 MAbs were found to be potentially specific for E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These MAbs recognized major immunogenic OMP band at 44kDa in Western blotting. Both MAbs belonged to the IgG1 isotype and recognized different epitopes of OMP as seen by competitive ELISA. These MAbs strongly reacted with all 17 isolates of E. tarda used in our study by indirect ELISA and Western blotting. Interestingly, no reaction was observed with the reference strain of E. tarda (MTCC 2400). The sensitivity of 3F10 MAb to detect whole cells of E. tarda was up to a level of 1x10(4)CFU/ml in indirect ELISA. No cross-reactivity of MAbs were seen with Escherichia coli, Salmonella arizonae, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae, Flavobacterium ferrugineum and Mycobacterium tuberculosis. These MAbs could be used for specific detection of E. tarda infection in fish by immunoassays.